Recombinant poxvirus expressing homologous genes inserted into the poxviral genome

ABSTRACT

The present invention relates to a recombinant poxvirus vector capable of expressing two or more homologous, foreign sequences, which derive from different variants of a microorganism, and which have a homology of 50% or above. The invention further relates to a method for preparing such recombinant poxvirus and the use of such recombinant poxvirus as medicament or vaccine. Additionally, a method for affecting preferably inducing, an immune response in a living animal, including a human, is provided.

The present invention relates to a recombinant poxvirus capable ofexpressing two or more homologous foreign genes. Said genes areheterologous to the viral genome, but homologous in comparison to eachother. The genes are especially derived from closely related variants orsubtypes of a microorganism. The invention further relates to a methodfor preparing such recombinant poxvirus and to the use of suchrecombinant poxvirus as medicament or vaccine. Additionally, a methodfor affecting, preferably inducing, an immune response in a livinganimal, including a human, is provided.

BACKGROUND OF THE INVENTION

Every living organism is constantly challenged by infectious orpathogenous agents such as bacteria, viruses, fungi or parasites. Theso-called immune system prevents the organism from permanent infections,diseases or intoxination caused by such agents.

The immune system of a mammal can be divided into a specific and anunspecific part although both parts are closely cross-linked. Theunspecific immune response enables an immediate defense against a widevariety of pathogenic substances or infectious agents. The specificimmune response is raised after a lag phase, when the organism ischallenged with a substance for the first time. This specific immuneresponse is mainly based on the production of antigen-specificantibodies and the generation of macrophages and lymphocytes, e.g.cytotoxic T-cells (CTL). The specific immune response is responsible forthe fact that an individual who recovers from a specific infection isprotected against this specific infection but still is susceptible forother infectious diseases. In general, a second infection with the sameor a very similar infectious agent causes much milder symptoms or nosymptoms at all. This so-called immunity persists for a long time, insome cases even lifelong. The underlying effect is often referred to asimmunological memory, which can be used for vaccination proposes.

With the term vaccination a method is described, where an individual ischallenged with a harmless, partial or inactivated form of theinfectious agent to affect, preferably induce, an immunological responsein said individual, which leads to long lasting—if not lifelong—immunityagainst the specific infectious agent.

The human smallpox disease is caused by Variola virus: Variola virusbelongs to the family of Poxyiridae, a large family of complex DNAviruses that replicate in the cytoplasma of vertebrate and invertebratecells.

The family of Poxyiridae can be divided into the two subfamiliesChordopoxyirinae and Entomopoxyirinae based on vertebrate and insecthost range. The Chordopoxyirinae comprise beside others the genera ofOrthopoxviruses and Avipoxviruses (Fields Virology, ed. by Fields B. N.,Lippincott-Raven Publishers, 3^(rd) edition 1996, ISBN: 0-7817-0253-4,Chapter 83).

The genera of Orthopoxviruses comprises variola virus, the causativeagent of human smallpox, and also other viruses with economicalimportance, e.g. camelpox, cowpox, sheeppox, goatpox, monkeypox andVaccinia virus. All members of this genus are genetically related andhave similar morphology or host range. Restriction endonuclease mapshave even shown high sequence identity from up to 90% between differentmembers of the Orthopoxviruses (Mackett & Archard, [1979], J Gen Virol,45: 683-701).

Vaccinia virus (VV) is the name given to the agent that was used atleast the last 100 years for the vaccination against smallpox. It is notknown whether VV is a new species derived from cowpox or variola virusby prolonged serial passages, the living representative of a now extinctvirus or maybe a product of genetic recombination. Additionally, incourse of the VV history, many strains of Vaccinia have arisen. Thesedifferent strains demonstrate varying immunogenicity and are implicatedto varying degrees with potential complications, the most serious ofwhich is post-vaccinial encephalitis. However, many of these strainswere used for the vaccination against smallpox. For example the strainsNYCBOH, Western Reserve or Wyeth were used primarily in US, while thestrain Ankara, Bern, Copenhagen, Lister and MVA were used forvaccination in Europe. As a result of the worldwide vaccination programwith these different strains of VV in 1980 the WHO finally declared thesuccessful eradication of variola virus.

Today, VV is mainly used as a laboratory strain, but beside this it isstill considered as the prototype of Orthopoxviruses, which is also thereason why VV became one of the most intensively characterized viruses(Fields Virology, ed. by Fields B. N., Lippincott-Raven Publishers,3^(rd) edition 1996, ISBN: 0-7817-0253-4, Chapter 83 and 84).

VV and more recently other poxviruses have been used for the insertionand expression of foreign genes. The basic technique for insertingforeign genes into live infectious poxvirus involves recombinationbetween pox DNA sequences flanking a foreign genetic element in a donorplasmid and homologous sequences present in the rescuing poxvirus.Genetic recombination is, in general, the exchange of homologoussections of DNA between two strands of DNA. In certain viruses RNA mayreplace DNA. Homologous sections of nucleic acid are sections of nucleicacid (DNA or RNA), which have the same sequence of nucleotide bases.Genetic recombination may take place naturally during the replication ormanufacture of new viral genomes within an infected host cell. Thus,genetic recombination between viral genes may occur during the viralreplication cycle that takes place in a host cell, which is co-infectedwith two or more different viruses or other genetic constructs. Asection of DNA from a first genome is used interchangeably inconstructing the section of the genome of a second co-infecting virus inwhich the DNA is homologous with that of the first viral genome.

Successful expression of the inserted DNA genetic sequence by themodified infectious virus requires two conditions. First, the insertionshould be into a nonessential region of the virus in order that themodified virus remains viable. The second condition for expression ofinserted DNA is the presence of a promoter in the proper relationship tothe inserted DNA. Regularly, the promoter is located upstream from theDNA sequence to be expressed.

The usefulness of recombinant VV expressing, e.g., Hepatitis B virussurface antigen (HBsAg), Influenza virus hemagglutinin (InfHA) orPlasmodium knowlesi sporozoite antigen, as live vaccines for theprophylaxis of infectious diseases has been demonstrated and reviewed(Smith, et al. Biotechnology and Genetic Engineering Reviews 2,383-407).

A further advantage of VV is the capacity to take up multiple foreignsequences, genes or antigens within a single VV genome (Smith & Moss[1983], Gene, 25(1): 21-28). Furthermore, it has been reported that itis possible to elicit immunity to a number of heterologous infectiousdiseases with a single inoculation of a polyvalent vaccine (Perkus etal., [1985], Science, Vol. 229, 981-984).

One example of the expression of various antigens by a single VV isdescribed by Bray et al. It was shown that a recombinant VV, which iscapable to express three different structural proteins of Dengue virusserotype 4, namely the capsid (C), pre-membrane (prM), envelope (E)protein, and two non-structural proteins of Dengue virus serotype 4,namely NS1 and NS2a, had the ability to protect mice against ahomologous Dengue virus serotype 4 challenge (Bray et al., [1989],Virology 2853-2856).

The Dengue virus with its four serotypes, Dengue virus serotype 1(Den-1) through Dengue virus serotype 4 (Den-4), is one important memberof the Flavivirus genus with respect to infections of humans. Denguevirus infection produces diseases that range from flu-like symptoms tosevere or fatal illness, Dengue haemorrhagic fever (DHF) with shocksyndrome (DSS). Dengue outbreaks continue to be a major public healthproblem in densely populated areas of the tropical and subtropicalregions, where mosquito vectors are abundant.

The concern over the spread of Dengue infection and other diseasesinduced by mosquito-borne Flaviviruses in many parts of the world hasresulted in more efforts being made towards the development of Denguevaccines, which could prevent both Dengue fever (DF), and Denguehaemorrhagic fever (DHF) and in vaccines useful to protect thevaccinated individual against infections induced by some or allmosquito-borne flaviviruses.

While most cases of DF are manifested after the first infection by anyof the four serotypes, a large percentage of DHF cases occur in subjectswho are infected for the second time by a serotype, which is differentfrom the first infecting serotype of Dengue virus. These observationsgive rise to the hypothesis that sequential infection of an individualhaving antibodies against one Dengue serotype by a different virusserotype at an appropriate interval may result in DHF in a certainnumber of cases.

Accordingly, vaccination against one serotype does not result in acomplete protection against Dengue virus infection, but only againstinfection with the same Dengue virus strain. Even more important, aperson vaccinated against one serotype, has an increased risk ofdeveloping severe complications such as Dengue haemorrhagic fever whensaid person is infected from a Dengue virus strain of a differentserotype.

Thus, a multivalent vaccine that contains antigens from all four Denguevirus serotypes is desired.

So far it had been suggested to prepare multivalent vaccines by mixing apanel of recombinant VV, each VV encoding sequences of a differentviruses (Moss, [1990] Immunology, 2, 317-327). However, such amultivalent vaccine comprises several disadvantages. Firstly, it iscumbersome to generate several independent recombinant VV. Beside theseparated production processes, also quality control and qualityassurance is highly time consuming. Secondly, an infection with amixture of recombinant viruses expressing different sequences alwaysbears the risk that the infection event is not particularly wellbalanced. The main risk is that only individual recombinants, but notall different recombinants comprised in the multivalent vaccine willinfect target cells. One reason might be an uneven distribution ofrecombinant viruses. Another reason might be interferences between thedifferent recombinant viruses while infecting single cells. Suchinterferences are known as phenomenon of superinfection. In this case,only some antigens, but not all different antigens of the multivalentvaccine will finally be expressed from infected cells and, thus,presented to the immune system of a patient. As a consequence, immuneprotection will be obtained only against some of the antigens, but isfar from providing a complete immune protection against the variousantigens presented or presentable by the multivalent vaccine.

In the context of a vaccine against Dengue virus infection the approachof a multivalent vaccine has the disadvantage that if the differentsequences are expressed in different amounts or in an unpredictablemanner, as it had been shown for the envelope protein of Dengue virus 2(Deuble et al., [1988], J. Virol 65: 2853), then such a vaccination ishighly risky for a patient. An incomplete vaccination using a panel ofrecombinant Vaccinia viruses will only provide an immune protectionagainst some, but not against all serotypes of Dengue virus.Unfortunately, in case of Dengue infection an incomplete vaccination isextremely unacceptable, since it increases the risk of lethalcomplications such as Dengue hemorrhagic fever.

OBJECT OF THE INVENTION

It is, therefore, an object of the present invention to provide astable, effective and reliable vaccine against infectious diseases,which can be caused by more than one strain, Glade, variant, subtype orserotype of said infectious disease causing microorganism.

It is a further object of the present invention to provide a stable,effective and reliable vaccine against Dengue virus infections, whichallows reliable vaccination against all Dengue virus serotypes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the idea to include into a poxvirushomologous genes derived from different strains, clades, variants,subtypes or serotypes of an infectious disease causing microorganism. Asalready mentioned above, there are, for example, 4 groups, subtypes orserotypes of Dengue virus existing which are all comprising the sametypes of genes as, e.g., the gene encoding the capsid (C) protein, thegene encoding the pre-membrane (PrM) or envelope (E) protein. However,the nucleic acid sequence of the same type of gene is not completelyidentical and not perfectly homologous, respectively, in all 4serotypes: For example, sequence comparison (with Lasergene 4.05Magalign, Macintosh) between the PrM genes of Dengue virus serotype 1,2, 3 and 4 (PrM1-4) revealed a sequence identity of 66.5-72.9%, i.e., ahomology of appr. 65-75%. It is assumed that differences and variations,respectively, in the genes of different subtypes of infectious diseasecausing microorganisms are the reasons why vaccination against onesubtype does not automatically result in protection against infectionswith other variants of the same microorganism. It was, therefore, theidea to generate a recombinant virus including closely related orhomologous genes derived from different strains, clades, variants,subtypes or serotypes of an infectious disease causing microorganism.However, as also already indicated above, homologous recombinationbetween homologous sequences occur during the viral life cycle and eventakes place between sections of DNA that are not perfectly homologous.It was, thus, expected that the insertion of homologous genes in asingle viral genome would result in homologous recombination and, thus,in deletions of the inserted homologous genes.

However, when generating a recombinant poxvirus comprising in its genomeat least two foreign genes with a homology of at least 60%, it wasunexpectedly found that said homologous genes remain stably insertedinto the viral genome.

Even if homologous genes, preferably with a homology of at least 50%,are inserted into different insertion sites of the viral genome, thegenes also remain stably inserted into the viral genome: In this case,it was expected that recombination events between said homologous geneswould additionally result in loss of viral genes important foramplification of the virus and for the viral life cycle, respectively,i.e., it was expected that the viral life cycle would seriously bedamaged. Since, additionally, the frequency of recombination isproportional to the distance between two linked genes, it was expectedthat frequency of recombination events between two or more homologousgenes located in different insertion sites would be high and, thus,result in deletions of said genes and/or in severe interferences.Accordingly, it was extremely surprising that no recombination eventsoccurred, but that the homologous genes remained stably inserted intothe different insertion sites of the viral genome.

According to the prior art, a recombinant poxvirus containing foreignDNA from flavivirus, such as Japanese Encaphalitis Virus (JEV), YellowFever Virus (YFV) and Dengue Virus, are known (U.S. Pat. No. 5,514,375).However, each gene derived from said flaviviruses were inserted only asingle time and into the same insertion site. Additionally, sequencecomparison with a suitable computer software (Lasergene 4.05 Megalign,Macintosh) revealed a homology of the genes inserted into the poxviralgenome and derived from JEV of 20.2%-29.6%, from YFV of 29.2%-45.3%, andfrom Dengue Virus of 22.8%-29.5%.

Similar disclosure applies to WO 98/13500 describing insertion of Denguevirus antigens into the same insertion site of Modified Vaccinia VirusAnkara (MVA), especially into deletion site II.

U.S. Pat. No. 5,338,683 describes insertion of gp 13 and 14 ofherpesvirus glycoprotein genes into two different insertion sites of asingle recombinant poxvirus; however, both genes have a homology of25.2% only.

Sequence comparison between influenza virus haemagglutinin andnucleoprotein gene inserted into the same insertion site (deletion siteIII) of a Modified Vaccinia Virus Ankara (MVA) resulted in a homology of49.1% (U.S. Pat. No. 5,676,950; Sutter et al., [1994], Vaccine 12:1032).

U.S. Pat. No. 5,891,442 discloses a recombinant poxvirus containing thecoding sequence for the polyprotein VP2, VP3 and VP4 of infectiousbursal disease. Said genes were fused and, thus, inserted into a singleinsertion site and have a homology of 41.9%-50.3%.

Finally, U.S. Pat. No. 6,217,882 describes a recombinant swinepox virusvector containing pseudorabies antigens gp50 and gp63 with a homology of52.7% inserted into the same insertion site.

In summary, it can be stated that according to the prior art homologousgenes or sequences having a homology of at least 50% are all insertedinto the same or a single insertion site within the viral genome.

According to the present invention, homologous genes or sequences have ahomology of at least 50%, i.e., a homology of 50%-100%, i.e., at least50% identical nucleotide bases. Genes or sequences having a homologybelow 50% can be considered as being heterologous. In the context of thepresent invention, the term “homologous” or “homology” is used whengenes or sequences are compared to each other, whereas the terms“foreign” gene, “exogenous” or “heterologous” sequence are used whengenes or sequences are compared to the poxviral genome; i.e., said termsrefer to a DNA sequence which is in nature not normally found associatedwith a poxvirus as used according to the invention. Accordingly, thepresent invention relates to a recombinant poxvirus comprising at leasttwo genes which are heterologous in comparison to the viral genome, butwhich are homologous among each other. The term “genes” refers to codingsequences, which encode, e.g., proteins, polypeptides, peptides,antigens and the like. Proteins, polypeptides or peptides translatedfrom homologous genes fulfill the same tasks and show the samefunctional properties. Homologous genes are regularly derived fromdifferent, but related sources or organisms. According to one embodimentof the present invention, the homology in the coding sequences ispreferably 70% to 80%, more preferably 80% to 90% or 90% to 100%. Mostpreferred is a homology of 65% to 75%.

Since the recombinant poxvirus according to the present inventioncomprises the relevant genetic information in one single infectious unitor in one virus particle only, there is no risk of uneven infection andunbalanced expression of the different homologous sequences. Thus, therecombinant poxvirus according to the present invention comprising andcapable of expressing several closely or even closest related genes oralmost identical sequences in one infected cell is particularlyadvantageous for the generation of multivalent vaccines.

This advantage is particularly interesting for the development ofvaccines against diseases, which can be caused by several closelyrelated strains or serotypes of a virus, like e.g. Dengue virus.Recombinant poxviruses comprising homologous genes of different Denguevirus serotypes are described in the Examples.

The homologous genes or sequences according to the present invention canbe derived from any microorganism, such as any virus except the vectorvirus, any bacterium, any fungus or parasite. Preferably, the homologousgenes or sequences are derived from an infectious or pathogenicmicroorganism and most preferably from different strains or clades,variants, subtypes or serotypes of said microorganism.

The terms “strain” or “clade” are technical terms, well known to thepractitioner, referring to the taxonomy of microorganisms. The taxonomicsystem classifies all so far characterised microorganisms into thehierarchic order of Families, Genera, species, strains (Fields Virology,ed. by Fields B. N., Lippincott-Raven Publishers, 4^(th) edition 2001).While the criteria for the members of Family is their phylogeneticrelationship, a Genera comprises all members which share commoncharacteristics, and a species is defined as a polythetic class thatconstitutes a replicating lineage and occupies a particular ecologicalniche. The term “strain” or “clade” describes a microorganism, i.e.virus, which shares the common characteristics, like basic morphology orgenome structure and organisation, but varies in biological properties,like host range, tissue tropism, geographic distribution, attenuation orpathogenicity. The term “variants” or “serotypes” further distinguishesbetween members of the same strain, also called subtypes, which showindividual infection spectra or antigenic properties due to minorgenomic variations.

According to a further embodiment of the present invention thehomologous genes or sequences are selected from viruses, preferablyviruses, which belong to the genera of Flaviviruses, such aspreferable—but not limited to—Dengue virus, West Nile virus or Japaneseencephalitis virus; which belong to the genera of Retroviruses, such aspreferable—but not limited to—Human Immunodeficiency Virus (HIV); whichbelong to the genera of Enteroviruses, such as preferable—but notlimited to—Hand, Foot and Mouth disease, EV71; which belong to thegenera of Rotaviruses or which belong to the genera of Orthomyxoviruses,such as preferable—but not limited to—Influenza virus. Most preferredare homologous genes derived from a Flavivirus.

According to a further preferred embodiment, the homologous genes areselected from Dengue virus genes, preferably C, NS1 and/or NS2, orpreferably E, more preferably PrM. Most preferred are homologous genesderived from different serotypes of the virus, wherein said genes may bederived from one, two, three or from all of the 4 Dengue virusserotypes.

According to still a further embodiment the homologous genes areselected from different HIV stains or clades. Preferably the homologousgenes are selected from the gag/pol coding sequence, more preferablyfrom the env coding sequence or further preferably from a combination ofstructural and/or regulatory HIV coding sequences.

The vector virus suitable for the present invention is selected from thegroup of poxviruses, which can be easily cultured in selected host cellsas, e.g., avian host cells, but which are highly replication deficientor actually not replicating in humans or human cells.

According to some preferred embodiments the poxvirus according to thepresent invention is selected from the group comprising canarypoxviruses (Plotkin et al. [1995] Dev Biol Stand. vol 84: pp 165-170.Taylor et al. [1995] Vaccine, Vol 13. No. 6: pp 539-549), fowlpoxviruses (Afonso et al. [2000] J Virol, pp 3815-3831. Fields Virology,ed. by Fields B. N., Lippincott-Raven Publishers, 4^(th) edition 2001,Chapter 85: page 2916), penguin pox viruses (Stannard et al. [1998] JGen Virol, 79, pp 1637-1649) or derivatives thereof. Since these virusesbelong to the genera of Avipoxviruses they can be easily cultured andamplified in avian cells. However, in mammalian or human cells they arereplication defective, which means that essentially no or nearly noinfectious progeny viruses are produced.

According to a further embodiment of the present invention a Vacciniaviruse, preferrably an attenuated Vaccinia virus, is used for thegeneration of recombinant poxviruses comprising two or more homologousgenes.

Although it is known that Vaccinia viruses (VV) may undergo homologousrecombination of short homologous sequences and thereby may deletehomologous sequences (Howley et al., [1996], Gene 172: 233-237) theinventors provide a recombinant Vaccinia virus including homologoussequences or genes stably inserted into its genome. This finding isparticularly unexpected, since according to Howely et al. already shortsequences from up to 300 base pairs (bp) were sufficient to inducegenomic rearrangement and deletion of homologous sequences in Vacciniavirus. The practitioner would, thus, expect that longer sequences wouldinduce recombination events with an even higher probability. However,according to the present invention even sequences comprising completehomologous genes can be stably inserted into the genome of Vacciniavirus.

One—not limiting—example of a vaccinia virus is the highly attenuatedand host range restricted Vaccinia strain, Modified Vaccinia Ankara(MVA) (Sutter, G. et al. [1994], Vaccine 12: 1032-40). MVA has beengenerated by about 570 serial passages on chicken embryo fibroblasts ofthe Ankara strain of Vaccinia virus (CVA) (for review see Mayr, A., etal. [1975], Infection 3, 6-14). As a consequence of these long-termpassages CVA deleted about 31 kilobases of its genomic sequence. Theresulting virus strain, MVA, was described as highly host cellrestricted (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 [1991]). Atypical MVA strain is MVA 575 that has been deposited at the EuropeanCollection of Animal Cell Cultures under the deposition number ECACCV00120707.

In another embodiment the MVA-Vero strain or a derivative thereof can beused according to the present invention. The strain MVA-Vero has beendeposited at the European Collection of Animal Cell Cultures under thedeposition number ECACC V99101431 and ECACC 01021411. The safety of theMVA-Vero is reflected by biological, chemical and physicalcharacteristics as described in the International Patent ApplicationPCT/EP01/02703. In comparison to normal MVA, MVA-Vero has one additionalgenomic deletion.

The term “derivatives” of a virus according to the present inventionrefers to progeny viruses showing the same characteristic features asthe parent virus but showing differences in one or more parts of itsgenome.

Still another embodiment according to the present invention uses MVA-BN.MVA-BN has been deposited at the European Collection of Animal CellCultures with the deposition number ECACC V00083008. By using MVA-BN ora derivative thereof a particular safe virus vaccine is generated, sinceit has been shown that the MVA-BN virus is an extremely high attenuatedvirus, derived from Modified Vaccinia Ankara virus. Therefore, in themost preferred embodiment, MVA-BN or derivatives thereof containing twoor more homologous genes according to the present invention is used asviral vector. The term “derivative of MVA-BN” describes a virus, whichhas the same functional characteristics compared to MVA-BN. The featuresof MVA-BN, the description of biological assays allowing the evaluationwhether an MVA is MVA-BN and a derivative thereof and methods allowingthe generation of MVA-BN or derivatives thereof are described in WO02/42480 (incorporated herein by reference). One easy way to examine afunctional characteristic of MVA-BN or derivatives thereof is itsattenuation and lack of replication in human HaCat cells.

In a recombinant poxvirus according to the present invention theexpression of the exogenous sequences is controlled preferably by apoxviral transcriptional control element, more preferably by an MVA,canary pox, fowl pox, or penguin pox transcriptional control element ormost preferably a Vaccinia virus promoter. Poxyiral transcriptionalcontrol elements according to the present invention comprise furthermoreevery transcriptional control element functional in a poxviral system.

The insertion of the exogenous sequences according to the presentinvention is preferably directed into a non-essential region of thevirus genome. Non-essential regions are e.g. loci or open reading frames(ORF) of poxviral genes, which are non-essential for the poxviral lifecycle. Also intergenic regions, which describe the space inbetween twoORF, are considered as non-essential regions according to the presentinvention. In another embodiment of the invention, the exogenoussequences are inserted at a naturally occurring deletion site of the MVAgenome (disclosed in PCT/EP96/02926 and incorporated herein byreference).

The orientation of the inserted DNA does not have an influence on thefunctionality or stability of the recombinant virus according thepresent invention.

Since the recombinant poxvirus according to the invention is highlygrowth restricted and, thus, highly attenuated, it is an ideal candidatefor the treatment of a wide range of mammals including humans and evenimmune-compromised humans. Hence, the present invention also provides apharmaceutical composition and also a vaccine for inducing an immuneresponse in a living animal body, including a human.

The pharmaceutical composition may generally include one or morepharmaceutically acceptable and/or approved carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Suchauxiliary substances can be water, saline, glycerol, ethanol, wetting oremulsifying agents, pH buffering substances, or the like. Suitablecarriers are typically large, slowly metabolized molecules such asproteins, polysaccharides, polylactic acids, polyglycollic acids,polymeric amino acids, amino acid copolymers, lipid aggregates, or thelike.

For the preparation of vaccines, the recombinant poxvirus according tothe invention is converted into a physiologically acceptable form. Thiscan be done based on the experience in the preparation of poxvirusvaccines used for vaccination against smallpox (as described by Stickl,H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392). For example, thepurified virus is stored at −80° C. with a titre of 5×10E8 TCID₅₀/mlformulated in about 10 mM Tris, 140 mM NaCl pH 7.4. For the preparationof vaccine shots, e.g., 10E2-10E8 particles of the virus are lyophilizedin 100 ml of phosphate-buffered saline (PBS) in the presence of 2%peptone and 1% human albumin in an ampoule, preferably a glass ampoule.Alternatively, the vaccine shots can be produced by stepwisefreeze-drying of the virus in a formulation. This formulation cancontain additional additives such as mannitol, dextran, sugar, glycine,lactose or polyvinylpyrrolidone or other aids such as antioxidants orinert gas, stabilizers or recombinant proteins (e.g. human serumalbumin) suitable for in vivo administration. The glass ampoule is thensealed and can be stored between 4° C. and room temperature for severalmonths. However, as long as no need exists the ampoule is storedpreferably at temperatures below −20° C.

For vaccination or therapy the lyophilisate can be dissolved in 0.1 to0.5 ml of an aqueous solution, preferably physiological saline or Trisbuffer, and administered either systemically or locally, i.e.parenteral, subcutaneous, intravenous, intramuscular, by scarificationor any other path of administration know to the skilled practitioner.The mode of administration, the dose and the number of administrationscan be optimized by those skilled in the art in a known manner. However,most commonly a patient is vaccinated with a second shot about one monthto six weeks after the first vaccination shot.

The recombinant virus according to the present invention is used for theintroduction of the exogenous coding sequences into a target cell. Theintroduction of an exogenous coding sequence into a target cell may beused to produce in vitro proteins, polypeptides, peptides, antigens andepitopes, respectively. Furthermore, the method for introduction of ahomologous or of a heterologous sequence into cells may be applied forin vitro and in vivo therapy. For in vitro therapy, isolated cells thathave been previously (ex vivo) infected with the recombinant poxvirusaccording to the invention are administered to the living animal bodyfor inducing an immune response. For in vivo therapy, the recombinantpoxvirus according to the invention is directly administered to theliving animal body for inducing an immune response. In this case, thecells surrounding the site of inoculation are directly infected in vivoby the virus or its recombinant according to the invention. Afterinfection, the cells synthesize the proteins, polypeptides, peptides orantigens, which are encoded in the exogenous coding sequences and,subsequently, present them or parts thereof on the cellular surface.Specialized cells of the immune system recognize the presentation ofsuch foreign proteins, polypeptides, peptides, antigens and epitopes andlaunch a specific immune response.

Methods to obtain recombinant poxviruses or to insert exogenous codingsequences into a poxviral genome are well known to the person skilled inthe art. Additionally, the method is described in the examples and canalso be deduced or completed from the following references:

-   Molecular Cloning, A laboratory Manual. Second Edition. By J.    Sambrook, B. F. Fritsch and T. Maniatis. Cold Spring Harbor    Laboratory Press. 1989: describes techniques and know how for    standard molecular biology techniques such as cloning of DNA, DNA    and RNA isolation, western blot analysis, RT-PCR and PCR    amplification techniques.-   Virology Methods Manual. Edited by Brian W J Mahy and Hillar O    Kangro. Academic Press. 1996: describes techniques for the handling    and manipulation of viruses.-   Molecular Virology: A Practical Approach. Edited by A J Davison and    R M Elliott. The Practical Approach Series. IRL Press at Oxford    University Press. Oxford 1993. Chapter 9: Expression of genes by    Vaccinia virus vectors.-   Current Protocols in Molecular Biology. Publisher: John Wiley and    Son Inc. 1998. Chapter 16, section IV: Expression of proteins in    mammalian cells using Vaccinia viral vector: describes techniques    and know-how for the handling, manipulation and genetic engineering    of MVA.

For the generation of recombinant poxviruses according to the presentinvention different methods may be applicable: The DNA sequence to beinserted into the virus may be placed into an E. coli plasmid constructinto which DNA homologous to a section of DNA of the poxvirus has beeninserted. Seperately, the DNA sequence to be inserted is ligated to apromoter. The promoter-gene linkage is positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of pox DNA containinga non-essential locus. The resulting plasmid construct is amplified bygrowth within E. coli bacteria and isolated. The isolated plasmidcontaining the DNA gene sequence to be inserted is transfected into acell culture, e.g., chicken embryo fibroblasts (CEFs), along with thepoxvirus. Recombination between homologous pox DNA in the plasmid andthe viral genome, respectively, gives a poxvirus modified by thepresence of foreign DNA sequences.

According to a more preferred embodiment, a cell of a suitable cellculture as, e.g., CEF cells, is infected with a poxvirus. The infectedcell is, subsequently, transfected with a first plasmid vectorcomprising the foreign gene, preferably under the transcriptionalcontrol of a poxvirus expression control element. As explained above,the plasmid vector also comprises sequences capable of directing theinsertion of the exogenous sequence into a selected part of the poxviralgenome. Optionally, the plasmid vector contains also a cassettecomprising a marker and/or selection gene operably linked to a poxviralpromoter. Suitable marker or selection genes are, e.g., the genesencoding the Green Fluorescent Protein, β-Galactosidase, neomycin,phosphoribosyltransferase or other markers. The use of selection ormarker cassettes simplifies the identification and isolation of thegenerated recombinant poxvirus. However, a recombinant poxvirus can alsobe identified by PCR technology. Subsequently, a further cell isinfected with the recombinant poxvirus obtained as described above andtransfected with a second vector comprising a gene, which is homologousto the gene included in the first vector. In case, this gene shall beincluded into a different insertion site of the poxviral genome, thesecond vector also differs in the sequence directing the integration ofthe homologous gene into the genome of the poxvirus. After homologousrecombination occurred, the recombinant virus comprising two homologousgenes can be isolated. For introducing more than two homologous genesinto the recombinant virus, the steps of infection and transfection arerepeated by using the recombinant virus isolated in previous steps forinfection and by using a further vector comprising a further homologousgene for transfection.

Alternatively, the steps of infection and transfection as describedabove are interchangeable, i.e., a suitable cell may at first betransfected by the plasmid vector comprising the foreign gene and, then,infected with the poxvirus.

As a further alternative, it is also possible to introduce eachhomologous gene into different viruses, coinfect a cell with all theobtained recombinant viruses and screen for a recombinant including allhomologous genes.

The invention further provides a kit comprising two or more plasmidvector constructs capable of directing the integration of expressable,homologous genes into the poxvirus genome. Beside a suitable cloningsite such plasmid vectors comprise sequences capable of directing theinsertion of the exogenous sequence to selected parts in the poxviralgenome. Optionally, such vectors comprise selection or marker genecassettes. The kit further comprises means and instructions to selectviruses, which are recombinant for one or several of the homologousgenes and optionally a selection or marker gene, inserted via saidvector constructs.

According to another further embodiment the invention includes DNAsequences or parts thereof derived from or homologous to the recombinantpoxvirus of the present invention. Such sequences comprise at least partof the exogenous sequence comprising at least a fragment of one of thehomologous gene according to the present invention and at least afragment of the genomic poxvirus sequence according to the presentinvention, said genomic poxvirus sequences preferably flanking theexogenous sequence.

Such DNA sequences can be used to identify or isolate the virus or itsderivatives, e.g. by using them to generate PCR-primers, hybridizationprobes or in array technologies.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Schematic presentation of the insertion sites of the four PrM(=pre-membrane) genes (serotype 1-4) in the MVA genome according toExample 1.

FIGS. 2-9 and 12-17: Insertion plasmid vector constructs indicating thename of the vector, its size and the localization of the sequences ofinterest such as: AmpR=ampicillin resistance gene, bfp=blue florescenceprotein gene, dA=deletion A, dE=deletion E, d2=deletion 2, Ecogpt=E.coli guanosinphosphoribosyl-transferase gene, EGFP=enhanced greenflorescence protein gene, F1=flanking sequence 1, F2=flanking sequence2, I4L=intergenic region I4L, IGR=intergenic region, NPT II=neomycinresistance gene, P=poxvirus promoter, pr7.5=Vaccinia promoter 7.5,PrN=pre-membrane gene of Dengue virus, number indicating from which ofthe four serotypes it derives, rpt=repetition of flanking sequence.

FIG. 10: PCR verification of the vector cloning strategies of fourdifferent insertion vectors (pBN49, pRN50, pRN40, pBN39). Each of theplasmids was tested with 4 different PCR primer combinations. Eachcombination is specific for one distinct PrM sequence integrated intoone distinct insertion site.

FIG. 11: PCR verification of the recombinant poxvirus including fourhomologous Dengue virus PrM genes (Example 1). While in the upper partof the gel the different PCR results of the recombinant virus are shown,the lower part provides the results of the same PCR reactions of thecontrol plasmids as indicated. The plasmids containing the homologoussequences are named pBN39, pBN49 or pBN50. PrM stands for the insertedpre-membrane genes of Dengue virus, wherein the numbers indicate fromwhich of the four serotypes it derives. dA=deletion A, dE=deletion E,d2=deletion 2, I4L=intergenic region I4L describes the insertion site ofthe exogenous DNA.

FIG. 18: Schematic presentation of the insertion sites of three PrMgenes (serotype 2-4) in the MVA genome according to Example 2.

FIG. 19: PCR verification of the recombinant poxvirus including threehomologous Dengue virus PrM genes inserted into intergenic regions(Example 2). The upper panel shows the results of the PCR reactionsspecific for PrM2; the middle panel shows the results of the PCRreactions specific for PrM3, and the lower panel shows the results ofthe PCR reactions specific for PrM4. Lane 8 shows the same PCR reactionsof the control plasmids. Lane 2 shows the empty vector control MVA. PrMstands for the inserted pre-membrane genes of Dengue virus, wherein thenumbers indicate from which of the four serotypes it derives.M=Molecular weight marker.

The following examples will further. illustrate the present invention.It will be well understood by a person skilled in the art that theprovided examples in no way may be interpreted in a way that limits theapplicability of the technology provided by the present invention tothis examples.

EXAMPLE 1 Insertion Vectors Insertion Vector for Deletion A

For the insertion of exogenous sequences into the MVA genome at theso-called deletion A or deletion 1 respectively, corresponding to thegenome position 7608-7609, a plasmid vector was constructed, whichcomprises about 600 by of the flanking sequences adjacent to thedeletion site A. To isolate the flanking sequences from the genomicMVA-BN DNA, suitable PCR primers can be designed with suitable computersoftware (DNAsis, Hitashi software engineering, San Bruno, USA). Suchprimers comprise extensions with restriction enzyme sites, which will beused to clone the flanking sequences into a vector plasmid. In betweenthese flanking sequences, a selection gene cassette is introduced, e.g aNPT II gene (neomycin resistance) under the transcriptional control of apoxviral promoter. Additionally, there is a cloning site for theinsertion of additional genes or exogenous sequences to be inserted intodeletion site A. One such vector construct according to the presentinvention is disclosed in FIG. 2 (pBNX10).

Insertion Vector for Deletion E

For the insertion of exogenous sequences into the MVA genome at theso-called deletion E or deletion 4, respectively, corresponding with thegenome position 170480-170481, a vector was constructed, which comprisesabout 600 by of the flanking sequences adjacent to the deletion site E.The vector is designed and constructed like described above. In betweenthe flanking sequences is located an EGFP gene (green fluorescenceprotein, Clonetech) under the transcriptional control of a poxviralpromoter. Additionally, there is a cloning site for the insertion ofadditional genes or sequences to be inserted into deletion site A. Onesuch vector construct according to the present invention is disclosed inFIG. 3 (pBNX32).

Insertion Vector for Deletion 2

For the insertion of exogenous sequences into the MVA genome at theso-called deletion 2, corresponding with the genome position20718-20719, a vector was constructed, which comprises about 600 by ofthe flanking sequences adjacent to the deletion site 2. The vector isdesigned and constructed like described above. In between the flankingsequences is located an hbfp gene (humanized blue fluorescing protein,Pavalkis G N et al.) under the transcriptional control of a poxviralpromoter. Additionally, there is a cloning site for the insertion ofadditional genes or sequences to be inserted into deletion site 2. Onesuch vector construct according to the present invention is disclosed inFIG. 4 (pBNX36).

Insertion Vector for Intergenetic Region, I4L

For the insertion of exogenous sequences in the intergenetic region,between the ORF I3L and I4L, corresponding to the genome position 56760,a vector was constructed, which comprises about 600 by of the flankingsequences adjacent to the intergenetic region at the I4L locus. Thevector is designed and constructed like described above. In between theflanking sequences is located an Ecogpt gene (or gpt stands forphosphoribosyl-transferase gene isolated from E. coli) under thetranscriptional control of a poxviral promoter. Additionally, there is acloning site for the insertion of additional genes or sequences to beinserted into the intergenetic region after the I4L ORF. One such vectorconstruct according to the present invention is disclosed in FIG. 5(pBNX39).

Construction of Recombinant Poxvirus Comprising Several Homologous GenesIntegrated in its Genome. Insertion Vectors

For the insertion of the four PrM genes of the four serotypes of Denguevirus in the MVA genome four independent recombination vectors wereused.

These vectors contain—as described in details above—sequences homologousto the MVA genome for targeting insertion by homologous recombination.Additionally each vector contains a selection- or reporter genecassette.

The PrM sequences of the four Dengue virus serotypes were syntheticallymade by oligo annealing and PCR amplification. The PrM sequences werecloned downstream of poxvirus promoter elements to form an expressioncassette. This expression cassette was, then, cloned into the cloningsite of the relevant insertion vector constructs.

As result, the insertion vector construct for deletion A contained thePrM gene of Dengue virus serotype 2 (FIG. 6—pBN39). The insertion vectorconstruct for deletion 2 contained the PrM gene of Dengue virus serotype1 (FIG. 7—pBN49). The insertion vector construct for intergenic regionI4L contained the PrM gene of Dengue virus serotype (FIG. 8—pBN50). Theinsertion vector construct for deletion E contained the PrM gene ofDengue virus serotype 4 (FIG. 9—pBN40).

PCR Verification of the Insertion Vectors

For verification of the cloning strategies, PCR assays were performed.For these PCR assays the selected primer pairs are a combination of aprimer specifically binding to the specific flanking sequence relativeto the insertion site and a second primer specifically binding to one ofthe highly homologous Dengue virus PrM genes.

The insertion vector for deletion A containing the PrM gene of Denguevirus serotype 2 was screened with the Primers oBN93(CGCGGATCCATGCTGAACATCTTGAACAGGAGACGCAGA. SEQ ID NO.: 1) and oBN477(CATGATAAGAGATTGTATCAG. SEQ ID NO.: 2).

The insertion vector for deletion 2 containing the PrM gene of Denguevirus serotype 1 was screened with the Primers oBN194(ATGTTGAACATAATGAACAGGAGGAAAAGATCTGTGACCATGCTCCTCATGCTGCTGCCCACAGCCCTGGCGTTCCATCT.SEQ ID NO.: 3) and oBN476 (GATTTTGCTATTCAGTGGACTGGATG. SEQ ID NO.: 4).

The insertion vector for intergenic region. I4L containing the PrM geneof Dengue virus serotype 3 was screened with the Primers oBN255(CCTTAATCGAATTCTCATGTCATGGATGGGGTAACCAGCATTAATAGT. SEQ ID NO.: 5) andoBN479 (GCTCCCATTCAATTCACATTGG. SEQ ID NO.: 6).

The insertion vector for deletion E containing the PrM gene of Denguevirus serotype 4 was screened with the Primers oBN210(ATCCCATTCCTGAATGTGGTGTTAAAGCTACTGAGCGCTTCTCTCGTCTCCGTTCTCCGCTCTGGGTGCATGTCCCATAC.SEQ ID NO.: 7) and oBN478 (GTACATGGATGATATAGATATG. SEQ ID NO.: 8).

PCR experiments are performed in a Thermal cycler GeneAmp 9700 (PerkinElmer) using the Taq DNA Polymerase Kit containing 10×PCR buffer, MgCl₂buffer and Taq DNA polymerase (Roche, Cat.no. 201205) or equivalent. Ingeneral the PCR reactions were prepared with a total reaction volume of50 μl containing 45 μl mastermix, the sample DNA and ddH₂O as required.The mastermix should be prepared with 30.75 μl DdH₂O, 5 μl 10× buffer, 1μl dNTP-mix (10 mM each), 2.5 μl of each primer (5 pmol/μl), 3 μl MgCl₂(25 mM) and 0.25 μl Taq-polymerase (5 U/μl).

The amplification was performed using the following programme:

1) Denaturation: 4 min 94° C. 2) 30 Cycles: Denaturation: 30 sec 94° C.Annealing: 30 sec 55° C. Elongation: 1-3 min 72° C. 3) Elongation 7 min72° C. 4) Store  4° C.

Based on the size of the inserted gene the elongation time should atleast be 1 min/kb.

The PCR results shown in FIG. 10 demonstrate the specifity of the primercombinations used for the single insertions.

The primer combination oBN194/oBN476 is specific for deletion 2 and PrM1as insert. The expected PCR fragment of plasmid pBN49 has a size of 678by (shown in lane 3, upper part of the gel).

The primer combination oBN255/oBN479 is specific for intergenic regionI4L and PrM3 as insert. The expected PCR fragment of plasmid pBN50 has asize of 825 by (shown in lane 9, upper part of the gel).

The primer combination oBN210/oBN478 is specific for deletion E and PrM4as insert. The expected PCR fragment of plasmid pBN40 has a size of 607by (shown in lane 5, lower part of the gel).

The primer combination oBN93/oBN477 is specific for deletion A and PrM2as insert. The expected PCR fragment of plasmid pBN39 has a size of 636by (shown in lane 11, lower part of the gel).

Generation of the Recombinant MVA Via Homologous Recombination

For expression of foreign genes by a recombinant MVA, these genes haveto be inserted into the viral genome by a process called homologousrecombination. For that purpose, the foreign gene of interest had beencloned into an insertion vector, as described above. This vector has tobe transfected after infection of cells with MVA-BN. The recombinationwill take place in the cellular cytoplasm of the infected andtransfected cells. With help of the selection and/or reporter cassette,which is also contained in the insertion vector, cells comprisingrecombinant viruses are identified and isolated.

Homologous Recombination

For homologous recombination, BHK (Baby hamster kidney) cells or CEF(primary chicken embryo fibroblasts) cells are seeded in 6 well platesusing DMEM (Dulbecco's Modified Eagles Medium, Gibco BRL)+10% fetal calfserum (FCS) or VP-SFM (Gibco BRL)+4 mmol/1 L-Glutamine for a serum freeproduction process.

Cells need to be still in the growing phase and, therefore, should reach60-80% confluence on the day of transfection. Cells were counted beforeseeding, as the number of cells has to be known for determination of themultiplicity of infection (moi) for infection.

For infection, the MVA stock is diluted in DMEM/FCS orVP-SFM/L-Glutamine so that 500 μl dilution contain an appropriate amountof virus that will give a moi of 0.01. Cells are assumed to be dividedonce after seeding. The medium is removed from cells and cells areinfected with 500 μl of diluted virus for 1 hour rocking at roomtemperature. The inoculum is removed and cells are washed withDMEM/VP-SFM. Infected cells are left in 1.6 ml DMEM/FCS andVP-SFM/L-Glutamine respectively while setting up transfection reaction(Qiagen Effectene Kit).

For transfection, the “Effectene” transfection kit (Qiagen) is used. Atransfection mix is prepared of 1-5 μg of linearized insertion vector(total amount for multiple transfection) with buffer EC to give a finalvolume of 150 μl. Add 8.0 μl Enhancer per μg DNA, vortex and incubate atroom temperature for 5 min. Then, 25 μl of Effectene are added per μgDNA after vortexing stock tube and the solution is mixed thoroughly byvortexing and incubated at room temperature for 10 min. 600 μl ofDMEM/FCS and VP-SFM/L-Glutamine respectively, are added, mixed andsubsequently, the whole transfection mix is added to the cells, whichare already covered with medium. Gently the dish is rocked to mix thetransfection reaction. Incubation takes place at 37° C. with 5% CO₂ overnight. The next day the medium is removed and replaced with freshDMEM/FCS or VP-SFM/L-Glutamine. Incubation is continued until day 3.

For harvesting, the cells are scraped into medium, then the cellsuspension is transferred to an adequate tube and frozen at −20° C. forshort-term storage or at −80° C. for long term storage.

Insertion of PrM4 into MVA

In a first round, cells were infected with MVA-BN according to theabove-described protocol and were additionally transfected withinsertion vector pBN40 containing the PrM gene of Dengue virus serotype4 and as reporter gene the EGPF gene. Since the transfected vectorcontains a reporter gene, EGFP, the synthesized protein is detectablelatest on day three in cells infected with a recombinant virus.Resulting recombinant viruses have to be purified by plaquepurification.

For plaque purification infected cells (fluorescing or stained) areisolated with a pipet tip, resuspended and aspirated in 200 μl PBS ormedium. Then a fresh culture dish containing about 10E6 cells isinfected with 100 μl of the resuspended plaques. After 48 h cells aretaken up in 300 μl PBS. DNA is extracted from suspension and screenedwith PCR analysis. A clone that shows the expected bands is chosen andfresh 6-well plates are infected with different amounts of this virus.Overlaying the wells with 1% agarose avoids further spreading of virus.After 48 h infected cells comprising a recombinant virus clone areisolated.

This procedure is repeated until no wild-type MVA-BN can be detected inthe PCR analysis.

After 4 rounds of plaque purification recombinant viruses, MVA-PrM4,were identified by PCR assays using a primer pair selectively amplifyingthe expected insertion (oBN210 and oBN478, as described above) and ascontrol a primer pair specifically recognizing the insertion sitedeletion E (oBN453: GTTGAAGGATTCACTTCCGTGGA, SEQ ID NO.: 9 and oBN454:GCATTCACAGATTCTATTGTGAGTC, SEQ ID NO.: 10)

Insertion of PrM2 into MVA-PrM4

Cells were infected with MVA-PrM4 according to the above describedprotocol and were additionally transfected with insertion vector pBN39containing the PrM gene of Dengue virus serotype 2 and as selection genethe NPT II, a neomycin resistance gene. For purification of recombinantMVA expressing an antibiotic resistance gene three rounds of virusamplification under selective conditions before plaque purification arerecommended. For neomycinphosphotransferase selection G418 is added tothe medium. G418 is a derivative of neomycin and inhibits theprotein-biosynthesis by interference with the action of the ribosomes.NPT gene activity inactivates G418 by phosphorylation.

After 16 rounds of plaque purification under neomycin selectionrecombinant viruses, MVA-PrM4/PrM2, were identified by PCR assays usinga primer pair selectively amplifying the expected insertion (oBN93 andoBN477, as described above) and as control a primer pair specificallyrecognizing the insertion site deletion A (oBN477: as described above)and oBN452: GTTTCATCAGAAATGACTCCATGAAA, SEQ ID NO.: 11). Additionallyalso insertion of PrM4 into deletion E is verified with the primerpairs: oBN210-oBN478 and oBN453-oBN454.

Insertion of PrM1 into META

In a first round, cells were infected with MVA-BN according to the abovedescribed protocol and were additionally transfected with insertionvector pBN49 containing the PrM gene of Dengue virus serotype 1 and asreporter gene the hbfp, the gene for humanized blue fluorescing protein.The synthesized hbfp protein is detectable on day three in cellsinfected with a recombinant virus. Resulting recombinant viruses werepurified by plaque purification.

After 10 rounds of plaque purification recombinant viruses, MVA-PrM1,were identified by PCR assays using a primer pair selectively amplifyingthe expected insertion (oBN194 and oBN476, as described above) and ascontrol a primer pair specifically recognizing the insertion sitedeletion 2 (oBN54: CGGGGTACCCGACGAACAAGGAACTGTAGCAGAGGCATC, SEQ ID NO.:12 and oBN56: AACTGCAGTTGTTCGTATGTCATAAATTCTTTAATTAT, SEQ ID NO.: 13)

Insertion of PrM3 into MVA

In a first round, cells were infected with MVA-BN according to the abovedescribed protocol and were additionally transfected with insertionvector pBN50 containing the PrM gene of Dengue virus serotype 3 and asreporter gene the Ecogpt gene (Ecogpt or shortened to gpt stands forphosphoribosyltransferase gene). Resulting recombinant viruses werepurified by 3 rounds of plaque purification underphosphribosyltransferase metabolism selection by addition of mycophenolcacid, xanthin and hypoxanthin. Mycophenolic acid (MPA) inhibits inosinemonophosphate dehydrogenase and results in blockage of purine synthesisand inhibition of viral replication in most cell lines. This blockagecan be overcome by expressing Ecogpt from a constitutive promoter andproviding the substrates xanthine and hypoxanthine.

Resulting recombinant viruses, MVA-PrM3, were identified by PCR assaysusing a primer pair selectively amplifying the expected insertion(oBN255 and oBN479, as described above) and as control a primer pairspecifically recognizing the insertion site I4L (oBN499:CAACTCTCTTCTTGATTACC, SEQ ID NO.: 14 and oBN500: CGATCAAAGTCAATCTATG;SEQ ID NO.: 15).

Coinfection of BTA-PrE1 and MVA-PrM3

The cells were infected with equal amounts of MVA-PrM1 and MVA-PrM3according to the above protocol. After 3 rounds of plaque purificationunder phosphribosyltransferase metabolism selection of blue fluorescingclones of recombinant viruses were analyzed by PCR using the primerpairs (oBN255 and oBN479. oBN499 and oBN500. oBN194 and oBN476. oBN54and oBN56 as described above). Resulting recombinant viruses weredesignated MVA-PrM1/PrM3.

Coinfection of MVA-PrM1/PrM3 and MVA-PrM2/PrM4

The cells were infected with equal amounts of MVA-PrM1/PrM3 andMVA-PrM2/PrM4 according to the above protocol. Plaque purification wasperformed under phosphribosyltransferase metabolism and neomycinselection. Recombinant viruses inducing a green and blue flourescencewere isolated and analyzed by PCR using the primer pairs (oBN255 andoBN479. oBN499 and oBN500. oBN194 and oBN476. oBN54 and oBN56. oBN93 andoBN477. oBN477 and oBN452. oBN210 and oBN478. oBN453 and oBN454 asdescribed above).

The PCR analysis of the recombinant virus. (Clone 20) comprising allfour PrM genes is shown in FIG. 11. While in the upper part of the gelthe different PCR results of the recombinant virus are shown, the lowerpart provides the results of the same PCR reactions of the controlplasmids (as indicated). Lane 1, 10 and 11 show a 1 kb and a 100 bpmolecular marker.

The primer combination oBN210/oBN478 is specific for deletion E and PrM4as insert. The expected PCR fragment of the recombinant virus and theplasmid pBN40 has a size of 607 by (shown in lane 2).

The primer combination oBN453/oBN454 is specific for deletion E. Theexpected PCR fragment of the recombinant virus is 2.7 kb, the expectedband of the wild-type virus 2.3 kb (shown in lane 3). Also in the upperpart of the gel a band specific for a wild-type virus can be identified.This means that the recombinant virus preparation is not yet completelyfree of wild-type virus. Further plaque purification is necessary.

The primer combination oBN93/oBN477 is specific for deletion A and PrM2as insert. The expected PCR fragment of the recombinant virus and theplasmid pBN39 has a size of 636 by (shown in lane 4).

The primer combination oBN477/oBN452 is specific for deletion A. Theexpected PCR fragment of the recombinant virus is 4.1 kb, the expectedband of the wild-type virus 2.7 kb (shown in lane 5). In the upper partof the gel a band specific for a wild-type virus can be identified.

The primer combination oBN255/oBN479 is specific for intergenic regionI4L and PrM3 as insert. The expected PCR fragment of the recombinantvirus and the plasmid pBN50 has a size of 825 by (shown in lane 6).

The primer combination oBN499/oBN500 is specific for the intergenicregion of I4L. The expected PCR fragment of the recombinant virus is 1.0kb, the expected band of the wild-type virus 0.3 kb (shown in lane 7).

The primer combination oBN194/oBN476 is specific for deletion 2 and PrM1as insert. The expected PCR fragment of the recombinant virus and theplasmid pBN49 has a size of 678 by (shown in lane 8).

The primer combination oBN54/oBN56 is specific for deletion 2. Theexpected PCR fragment of the recombinant virus is 1.6 kb, the expectedband of the wild-type virus 0.9 kb (shown in lane 9). In the upper partof the gel a band specific for a wild-type virus can be identified.

Alternatively, one can produce 4 different viruses, coinfect cells withall four viruses and screen for a recombinant.

Improvements can also be achieved with recombination vectors, whichcontain further selection- or resistance markers.

EXAMPLE 2 Insertion Vectors Recombination Vector for Intergenic Region136-137 (IGR 136-137)

For the insertion of exogenous sequences into the MVA genome at theso-called intergenic region (IGR) 136-137 corresponding to the genomeposition 129.940 a plasmid vector was constructed which comprises about600 by of the flanking sequences adjacent to the insertion site. Toisolate the flanking sequences from the genomic MVA-BN DNA suitable PCRprimers can be designed. Such primers comprise extensions withrestriction enzyme sites, which will be used to clone the flankingsequences into a vector plasmid. In between this flanking sequences, aselection gene cassette is introduced, e.g. NPT II gene (neomycineresistance) under the transcriptional control of a poxviral promoter(P). Additionally there is a cloning site for the insertion ofadditional genes or exogenous sequences to be inserted into IGR 136-137(PacI). One such vector construct according to the present invention isdisclosed in FIG. 12 (pBNX67).

Recombination Vector for Intergenic Region 07-08 (IGR 07-08)

For the insertion of exogenous sequences into the MVA genome in theintergenic region (IGR) 07-08 corresponding to the genome position12.800, a plasmid vector was constructed which comprises about 600 by ofthe flanking sequences adjacent to the insertion site. To isolate theflanking sequences from the genomic MVA-BN DNA suitable PCR primers canbe designed. Such primers comprise extensions with restriction enzymesites, which will be used to clone the flanking sequences into a vectorplasmid. In between this flanking sequences, a selection gene cassetteis introduced, e.g., Ecogpt gene (Guanin-Phosphoribosyltransferase)under the transcriptional control of a poxviral promoter (P).Additionally, there is a cloning site for the insertion of additionalgenes or exogenous sequences to be inserted into IGR 07-08 (PacI). Onesuch vector construct according to the present invention is disclosed inFIG. 13 (pBNX88).

Recombination Vector for Intergenic Region 44-45 (IGR 44-45)

For the insertion of exogenous sequences into the MVA genome at theintergenic region (IGR) 44-45 corresponding to the genome position37.330, a plasmid vector was constructed which comprises about 600 by ofthe flanking sequences adjacent to the insertion site. To isolate theflanking sequences from the genomic MVA-BN DNA suitable PCR primers canbe designed. Such primers comprise extensions with restriction enzymesites, which will be used to clone the flanking sequences into a vectorplasmid. In between this flanking sequences, a selection gene cassetteis introduced, e.g., NPT II gene (neomycine resistance) under thetranscriptional control of a poxviral promoter (P). Additionally thereis a cloning site for the insertion of additional genes or exogenoussequences to be inserted into IGR 44-45 (PacI). One such vectorconstruct according to the present invention is disclosed in FIG. 14(pBNX87).

Construction of Recombinant Poxvirus Comprising Several Homologous GenesIntegrated in its Genome Insertion Vectors

For the insertion of the three PrM genes of serotype 2, 3 and 4 ofDengue virus in the MVA genome, three independent recombination vectorswere used.

These vectors contain—as described in detail above—sequences homologousto the MVA genome for targeting insertion by homologous recombination.Additionally each vector contains a selection and reporter genecassette.

The PrM sequences of three Dengue virus serotypes were syntheticallymade as described in Example 1.

As result, the insertion vector construct for IGR136-137 contained thePrM of Dengue virus serotype 4 (FIG. 15—pBN27). The insertion vectorconstruct for IGR 07-08 contained the PrM of Dengue virus serotype 2(FIG. 16—pBN34), and the insertion vector construct for IGR 44-45contained the PrM of Dengue virus serotype 3 (FIG. 17—pBN47).

Generation of the Recombinant MVA Via Homologous Recombination

The generation of recombinant MVA by homologous recombination wasperformed as described in Example 1.

The insertion sites for the PrM4, PrM3 and PrM 2 in the MVA genome aredepicted in FIG. 18.

Insertion of PrM 4 into EVA

In a first round, cells were infected with MVA-BN according to theabove-described protocol and were additionally transfected with theinsertion vector pBN27 containing the PrM gene of Dengue virus serotype4 and as a reporter gene the EGFP gene. Since the transfected vectorcontains a reporter gene, EGFP, the synthesized protein is detectablelatest on day three in cells infected with a recombinant virus.Resulting recombinant viruses have to be purified by plaque purificationas described in Example 1. After four rounds of plaque purificationrecombinant viruses, MVA-PrM4, were identified by PCR assays using aprimer pair selectively amplifying the insertion site IGR136-137(oBN1008: gataccgatcacgttcta. SEQ ID NO.: 16; and oBN1009ggatatgattatgtagag. SEQ ID NO.: 17).

Insertion of PrM 2 into MVA

Cells were infected with MVA-PrM4 according to the above-describedprotocol and were additionally. transfected with the insertion vectorpBN34 containing the PrM gene of Dengue virus serotype 2 and as areporter gene the BFP gene. Since the transfected vector contains areporter gene, BFP, the synthesized protein is detectable latest on daythree in cells infected with a recombinant virus. Resulting recombinantviruses have to be purified by plaque purification as described inExample 1. After six rounds of plaque purification recombinant virusMVA-PrM4+PrM2 was further passaged and amplified and a crude stock wasprepared. The recombinant was identified by PCR assays using a primerpair selectively amplifying the insertion site IGR07-08 (oBN 903:ctggataaatacgaggacgtg. SEQ ID NO.: 18; and oBN904: gacaattatccgacgcaccg;SEQ ID NO.: 19).

Insertion of PrM 3 into ETA

Cells were infected with MVA-PrM2+4 according to the above-describedprotocol and were additionally transfected with the insertion vectorpBN47 containing the PrM gene of Dengue virus serotype 3 and as areporter gene the EGFP gene. Since the transfected vector contains areporter gene, EGFP, the synthesized protein is detectable latest on daythree in cells infected with a recombinant virus. Resulting recombinantviruses have to be purified by plaque purification as described inExample 1. After three rounds of plaque purification recombinantviruses, MVA-PrM4+3+2, were identified by PCR assays using a primer pairselectively amplifying the insertion site IGR44-45 (oBN904:cgttagacaacacaccgacgatgg. SEQ ID NO.: 20; and oBN905cggatgaaaaatttttggaag. SEQ ID NO.: 21).

The PCR analysis of the recombinant virus comprising the three Denguevirus PrM genes is shown in FIG. 19. PCR experiments are performed asdescribed in Example 1. The primer combination oBN1008 and oBN1009 isspecific for IGR136-137, which contains the PrM4 insertion (FIG. 19,lower panel). The expected PCR fragment of the recombinant virus has asize of 1 kb (shown in lane 4, 5 and 6) as the plasmid positive control(lane 8). The empty vector control, devoid of PrM 4 shows the expectedfragment of 190 by (lane 2). Lane M shows the molecular weight markerand lanes 1, 3 and 7 are empty. The primer combination oBN902 and oBN903is specific for IGR07-08, which contains the PrM2 insertion (FIG. 19,upper panel). The expected PCR fragment of the recombinant virus has asize of 960 bp (shown in lane 4-6) as the plasmid positive control (lane8). The empty vector control, devoid of PrM 2 shows the expectedfragment of 190 by (lane 2). The primer combination oBN904 and oBN905 isspecific for IGR44-45, which contains the PrM3 insertion (FIG. 19,middle panel). The expected PCR fragment of the recombinant virus has asize of 932 bp (shown in lane 4-6) as the plasmid positive control (lane8). The empty vector control, devoid of PrM 2 shows the expectedfragment of 185 by (lane 2).

1-27. (canceled)
 28. A recombinant Modified Vaccinia Virus Ankara (MVA)vector comprising at least two homologous foreign nucleotide sequencesthat are at least 50% homologous in comparison to each other, whereineach of the foreign nucleotide sequences is inserted into a differentsite in the MVA genome, and wherein each of the foreign nucleotidesequences is stably expressed in an infected host cell.
 29. Therecombinant MVA vector of claim 28, wherein the recombinant MVA vectorcomprises at least two homologous foreign nucleotide sequences that areat least 60% homologous in comparison to each other.
 30. The recombinantMVA vector of claim 28, wherein the recombinant MVA vector comprises atleast two homologous foreign nucleotide sequences that are 50-60%homologous in comparison to each other.
 31. The recombinant MVA vectorof claim 28, wherein the recombinant MVA vector comprises at least twohomologous foreign nucleotide sequences that are 60-75% homologous incomparison to each other.
 32. The recombinant MVA vector of claim 28,wherein the recombinant MVA vector comprises at least two homologousforeign nucleotide sequences that are 65-75% homologous in comparison toeach other.
 33. The recombinant MVA vector of claim 28, wherein at leastone of the foreign nucleotide sequences is inserted into an intergenicregion.
 34. The recombinant MVA vector of claim 33, wherein theintergenic region is IGR44/45.
 35. The recombinant MVA vector of claim33, wherein the intergenic region is IGR136/137.
 36. The recombinant MVAvector of claim 33, wherein the intergenic region is IGR07/08.
 37. Therecombinant MVA vector of claim 33, wherein the intergenic region is IGRI4L.
 38. The recombinant MVA vector of claim 28, wherein at least one ofthe foreign nucleotide sequences is inserted into a naturally-occurringdeletion site.
 39. The recombinant MVA vector of claim 38, wherein thenaturally-occurring deletion site is deletion site
 1. 40. Therecombinant MVA vector of claim 38, wherein the naturally-occurringdeletion site is deletion site
 2. 41. The recombinant MVA vector ofclaim 38, wherein the naturally-occurring deletion site is deletion site4.
 42. The recombinant MVA vector of claim 28, wherein the recombinantMVA vector comprises at least two homologous foreign nucleotidesequences of greater than 300 nucleotides.
 43. The recombinant MVAvector of claim 28, wherein the homologous foreign nucleotide sequencesencode viral antigens.
 44. The recombinant MVA vector of claim 43,wherein the homologous foreign nucleotide sequences encode Flavivirusantigens.
 45. The recombinant MVA vector of claim 28, wherein thehomologous foreign nucleotide sequences encode bacterial antigens. 46.The recombinant MVA vector of claim 28, wherein the homologous foreignnucleotide sequences encode fungal antigens.
 47. The recombinant MVAvector of claim 28, wherein the homologous foreign nucleotide sequencesencode parasite antigens.
 48. The recombinant MVA vector of claim 28,wherein the recombinant MVA vector contains three or less homologousforeign nucleotide sequences.
 49. The recombinant MVA vector of claim28, wherein the recombinant MVA vector comprises at least two homologousforeign nucleotide sequences that are identical.
 50. The recombinant MVAvector of claim 49, wherein the two homologous foreign nucleotidesequences are promoter elements.